The present invention is directed to a Lef1/β-catenin-dependent reporter and to transgenic fish containing this reporter. The present invention is also directed to the use of the reporter and the transgenic fish as a model for the β-catenin signaling pathway. The model is useful for identifying genes in the β-catenin signaling pathway and for identifying drugs that can modulate the β-catenin signaling pathway. Such drugs are useful for treating or preventing melanoma, colorectal cancer, and osteoporosis, among other disease conditions.
The publications and other materials used herein to illuminate the background of the invention, and in particular, cases to provide additional details respecting the practice, are incorporated by reference, and for convenience are referenced in the following text by author and date and are listed alphabetically by author in the appended bibliography.
Secreted Wnt ligands activate receptor-mediated signal transduction pathways, resulting in changes in gene expression, cell behavior, cell adhesion, and cell polarity. Investigations of these pathways have been driven for two decades by the knowledge that Wnt signaling is involved in both embryonic development and cancer. This knowledge has fostered a rigorous scientific dissection of Wnt signaling on the basis of genetic studies in the mouse Mus musculus, the fruit fly Drosophila melanogaster, the nematode Caenorhabditis elegans, and the zebrafish Danio rerio, as well as cell biological and biochemical studies in mammalian cultured cells and the frog Xenopus laevis. This worldwide effort has established that multiple Wnt signaling pathways are activated by a multigene family of Wnt ligands.
The first Wnt pathway to be discovered, and the best understood, is the canonical Wnt pathway that activates the function of β-catenin shown in FIG. 2, with more components, interactions, and target genes described in the canonical STKE Connections Map Wnt/β-catenin Pathway (Moon, 2002a). Acting through a core set of proteins that are highly conserved in evolution, this pathway regulates the ability of β-catenin to activate transcription of specific target genes. This regulation, in turn, results in changes in expression of genes that modulate cell fate, proliferation, and apoptosis. Components of the β-catenin signaling pathway are also regulated by other signals (FIG. 2), promoting interest in understanding how Wnts can function in combination with other signaling pathways. As more signaling pathways are added to the STKE Connections Maps, it will be possible for both casual users and experts to better understand and predict the outcome of increasingly complex combinatorial signaling.
Activation of the Wnt/β-catenin signaling pathway holds both promise and perils for human medicine. The perils have been known for some time—activation of this signaling pathway through loss-of-function mutations in the tumor suppressors adenomatous polyposis coli (APC) protein and axin, or through gain-of-function mutations in β-catenin itself, are linked to diverse human cancers, including colorectal cancers and melanomas (Polakis, 2000). This connection has fueled a search for Wnt/β-catenin pathway antagonists, which may become lead compounds for anticancer drugs. Greater knowledge of the Wnt/β-catenin pathway may benefit patients with other diseases and conditions, because this pathway is involved in regulating angiogenesis (Ishikawa et al., 2001; Wright et al., 1999), adipogenesis (Ross et al., 2000), and stem cell proliferation (Taipale and Beachy, 2001). For example, in the area of bone density, loss of function of a Wnt/β-catenin pathway co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), results in low bone mass in children and heterozygous parents (Gong et al., 2001). Conversely, apparent gain-of-function mutations in the same gene result in an autosomal dominant high bone-mass trait (Little et al., 2002). Thus, both antagonists and agonists of components of the Wnt/β-catenin pathway may prove therapeutic in cancer and in stimulating cell and bone replacement, respectively.
Given the clear link between the Wnt/β-catenin signaling pathway and human diseases, and the conservation of molecular functions across many animal taxa, understanding the mechanisms of Wnt signaling benefit substantially from studies in model systems. The specific pathways in the STKE Connections Maps help to promote the uses of model organisms to understand Wnt/β-catenin signaling. Currently, pathways in Drosophila (Boutros and Perriman, 2002), C. elegans (Bowerman, 2002a; Bowerman, 2002b; Bowerman, 2002c; Bowerman, 2002d) and Xenopus (Moon 2002b) are available, with future additions to include pathways for mouse, chicken, and zebrafish. Supporting this goal of including pathways from more species, much of the earliest work on Wnt signaling and its effects on adhesion and the cytoskeleton was conducted on mammalian cells in culture (Hinck et al., 1994), and subsequent work on the mouse has led to numerous discoveries, including the roles of Wnts as mitogens in the nervous system (Megason and McMahon, 2002), and as essential signaling factors in formation of the limbs (Martin, 2001), kidneys (Kispert et al., 1998), and female reproductive system (Heikkila et al., 2001). For a further review of Wnt pathway studies, see Moon et al. (2002).
The best characterized cellular output of Wnt/β-catenin signaling is the transcriptional activation of downstream target genes. Following Wnt pathway activation, cytoplasmic β-catenin accumulates and enters the nucleus, where it interacts with the Lef/Tcf class of transcription factors (Eastman and Grossched1, 1999; Sharpe et al., 2001). In zebrafish, two members of this family of HMG box proteins, Lef1 and Tcf3 (Headless, Hdl), have been implicated in early development (Dorsky et al., 1999; Pelegri and Maischein, 1998; Kim et al., 2000). Lef1 has been shown to act as a β-catenin-dependent transcriptional activator through its interactions with other coactivator molecules (Billin et al., 2000). Tcf3 is a transcriptional repressor in the absence of β-catenin (Brannon et al., 1999; Roose et al., 1998). Upon β-catenin binding, Tcf3-mediated repression is relieved by an unknown mechanism. Both proteins bind to similar upstream regulatory DNA sequences, termed Lef binding sites (Waterman et al., 1991).
Analysis of a headless mutation in zebrafish has suggested that the main role of this gene during development is to repress downstream targets in the forebrain (Kim et al., 2000), in part because mutant embryos can be rescued by expression of a form of Tcf3 that does not bind β-catenin. Other potential Tcf3 targets in Xenopus, such as siamois, require Lef binding sites only for their repression, and not for activation (Brannon et al., 1997). The question has therefore arisen of whether Tcf3 proteins ever act as gene activators in vivo or only as repressors that can be inactivated by Wnt signaling.
Although Wnts are expressed throughout the developing embryo, the range of Wnt signaling in vivo has been difficult to determine. As a result, the cell populations and target genes that respond to Wnt/β-catenin signals during development and in disease conditions are unidentified. In order to understand the multiple roles played by Wnt/β-catenin signaling, it is important to identify these very cell populations and genes. The CNS has remained particularly unexplored with respect to Wnt targets, considering that it was the first region to be identified as expressing a vertebrate wnt gene (Wilkinson et al., 1987) and has been subsequently shown to express numerous other Wnts as well (Hollyday et al., 1995). Overexpression and loss-of-function studies have suggested roles for Wnts throughout the CNS (Dickinson et al., 1995; Ikeya et al., 1997.
Thus, it is desired to develop model systems that can be used to (a) identify genes that modulate the β-catenin signaling pathway, (b) studying the relationship between the β-catenin signaling pathway and disease conditions, such as melanoma, colorectal cancer and osteoporosis among others, and (c) screen compounds to identify drugs that can modulate the β-catenin signaling pathway.